Voltage-gated ion channels are specialized integral membrane proteins that play a central role in a number of physiologic processes ranging from propagation of information in the nervous system to impulse generation in the heart. Studies of the gating of these channels reveal that a sizable quantity of charge moves across the membrane during the transition from the closed to open state. The mechanism by which this charge movement occurs is unclear yet its magnitude suggests that it must somehow be associated with a large physical movement of a segment of these channels. This proposal describes an effort to demonstrate such a conformational change in the Drosophila Shaker K+ channel using a combination of molecular biological, biochemical, and electrophysiological techniques. Site-directed mutagenesis and the Xenopus oocyte expression system will be used to introduce single cysteines into the K+ channel sequence, creating potential targets for cysteine-specific biotinylation. Under electrophysiologic recording, oocytes with open channels will be exposed to a biotinylating reagent and then to streptavidin, a large molecule that binds tightly to biotin. If the introduced cysteine is in a region of the channel that accesses the extracellular space during opening, such exposure should generate a large complex that will lock the channel into this open state. By varying the location of the introduced cysteine, regions of the channel that translocate can be identified. This physical movement will be correlated with charge movement by measuring, in a novel way, the gating charge present in single mutant channels. The long term objective of this project is to provide further insight into the structure and function of voltage-gated channels. Such information is likely to. shed light on several physiologic and pathophysiologic processes and should be useful in the design of therapeutic agents used in the treatment of such conditions as heart failure, arrhythmias and hypertension. This award will be instrumental in preparing the candidate for a career as a basic scientist and clinician studying ion channel structure and function and bringing this knowledge to bear on some of the pathophysiologic processes with which these channels are associated. Of particular importance will be the acquisition of several tools new to the candidate to supplement his previous biophysical training. These include site-directed mutagenesis the Xenopus oocyte expression system and the techniques of patch clamp and two-microelectrode voltage clamp recording. Dr. MacKinnon is an expert at applying these techniques to questions of ion channel function and is committed to the candidate's development into an independent investigator. The location of his laboratory in the Department of Neurobiology affords exposure to the rich intellectual atmosphere of Harvard Medical School and its environs. These factors make Dr. MacKinnon an ideal sponsor.